Carotenoids are the most numerous and widespread group of pigments to be found in nature. Some particular examples of carotenoid compounds are: β-carotene, β-apo-4′-carotenal, β-apo-8′-carotenal, β-apo-12′-carotenal, β-apo-8′-carotenic acid, astaxanthin, canthaxanthin, zeaxanthin, cryptoxanthin, citranaxanthin, lutein, lycopene, torularodin-aldehyde, torularodin-ethylester, neurosporaxanthin-ethylester, zeta-carotene and dehydroplectaniaxanthin. Moreover, the carotenoid is known as one of the pigments that are widely used for colouring of food, cosmetics, medicines, and the like.
Carotenoid crystals are usually produced by a conventional chemical process. However, now-a-days there is a substantial demand for products derived from natural sources. When derived from a natural source, the carotenoid mostly is in the form of an oily extract (palm oil, algae oil). Although it is also possible to obtain a crystalline carotenoid, for instance, carotene, from natural sources, such as vegetables (for example carrots) or microorganisms (for example algae (Dunaliella) or fungi (Blakeslea)), the currently available processes to obtain relatively pure crystals from said natural sources have important disadvantages.
Isolation of crystalline carotenoid, such as β-carotene, from natural sources comprises for instance extraction of the β-carotene from said source, such as mentioned in international patent application WO 9803480, and additional purification steps. The extraction is carried out with various organic solvents, such as acetone, ethyl acetate, butyl acetate, hexane, dichloromethane or hexane, vegetable oils, or supercritical fluids, such as propane, ethylene or carbon dioxide. To obtain a relatively pure β-carotene preparation, a further purification of the extract is necessary. Several purification processes have been described, among which chromatography, adsorption/desorption processes and crystallization or precipitation.
Carotenoid crystals which are crystallized from the extract obtained after solvent extraction of a suitable natural source, for example by evaporation of the solvent, have an odor peculiar to the starting material and typically contain some impurities, for instance the solvent itself and impurities originating from the extraction step. In such cases recrystallizations are required, for instance as described in U.S. Pat. No. 3,268,606 and U.S. Pat. No. 4,439,629. The main drawback of recrystallization is that a large amount of solvent is required to solubilize the carotene. In addition, to recrystallize the carotene with a sufficiently high yield, large amounts of anti-solvent (precipitating solvent) are necessary as well. Thus, these processes have the disadvantage that large amounts of solvents are required and a considerable loss of carotene can easily occur. Moreover, solvent will be enclosed within the crystal lattices of the crystalline carotenoid.
In international patent application WO 9850574, a process for the isolation of a crystalline carotenoid compound from microbial biomass has been described which comprises the steps of disrupting the microbial cell walls and separating cellular debris from the carotenoid containing residue, including a wash of the microbial biomass. The disrupted cell mass or the carotenoid containing residue is treated with a solvent suitable to remove lipid and suspending the obtained carotenoid crystals in water to float the crystals. Thereafter, the crystalline product is separated and, optionally, further purified. In the international patent applications WO 9843620, WO 9723436 and WO 9731894, a process for the preparation of carotenoid compound from oleoresins has been described, the process comprising a treatment with an alkaline reagent in an organic medium and then addition of an anti-solvent to obtain the crystalline carotenoid compound. The main disadvantage of both the dissolution of the disrupted cell mass in a solvent and the addition of an anti-solvent is the inclusion of solvent into the carotenoid crystals.
Surprisingly, we have found that very pure carotenoid crystals substantially free of solvent in the crystal lattice can efficiently be isolated from a crystalline suspension from a microbial source without the use of extraction/anti-solvent processes, yielding carotenoid crystals with a purity of at least 95%.